Because enzymes can catalyze chemical transformations so effectively, there is increasing emphasis on the use of enzyme reactions in commercial processes. The relatively high cost of enzymes demands their reuse. Typically, if the reaction is performed under homogeneous conditions recovery of enzyme is difficult and expensive, which effectively precludes homogeneous enzymatic catalysis. The solution to this problem is to insolubilizer enzyme under conditions where a substantial portion of the enzymatic activity exhibited in solution remains under heterogenous reaction conditions.
One particular solution to the aforementioned problem is the construction of immobilized enzyme systems. An immobilized enzyme system consists of a support matrix to which there is bound an enzyme. A support matrix is a structure characterized as having good physical integrity and favorable properties toward liquid flow under conditions experienced in fixed bed reactors, and further characterized by having the ability to bind or immobilize enzymes with minimum perturbation of enzymatic action. By an immobilized enzyme system is meant the structure which results from immobilization of an enzyme on a support matrix.
The binding or immobilization of enzymes to a support matrix is represented by the extremes of physical and chemical binding forces. It is to be recognized that in most cases enzyme immobilization arises from a combination of such binding forces, although often one such force predominates, with the nature of enzyme immobilization generally being determined by the nature of the support matrix. As an example, when the support matrix is a resin, such as one of the phenol-formaldehyde type, binding is predominantly through physical forces. A similar result is obtained when the support matrix is of an ion exchange type. Where the support matrix is comprised of refractory inorganic material, such as inorganic oxides, glass, and ceramics, bearing or impregnated with organic material, for example, polyamines, either bearing pendant functional groups themselves or cross-linked with a bifunctional reagent which provides pendant functional groups, enzyme immobilization arises mainly by chemical reaction of a site on the enzyme with the pendant functional group so as to form a covalent bond. In such an instance binding is, at least predominantly, by chemical means.
Recently Cabral and co-workers, Biotechnology and Bioengineering, 23, 2083 (1981) described metal-link-activated inorganic supports as support matrices, especially controlled-pore glass treated with titanium tetrachloride, subsequently dried in air, then reacted with hexamethylenediamine followed by glutaraldehyde. Although the support matrices described herein are superficially similar, they are operationally different and distinct from the prior art matrices, as shown by direct comparison with the support matrix prepared according to Cabral et. al.
The object of this invention is to prepare support matrices for immobilized enzymes and other reactive entities, and to prepare immobilized enzyme systems thereform. An embodiment is a method comprising contacting a porous inorganic refractory oxide with a titanium tetrahalide, removing excess and unreacted titanium tetrahalide by means including heat in an inert atmosphere at between about 80.degree. and 200.degree. C., contacting the resulting titanated alumina with a diamine or other polyamine, removing unreacted amine, contacting the mass with a bifunctional monomer, and recovering the resulting support matrix.
The purpose of the method described is to react surface hydroxyl groups from a porous refractory inorganic oxide with a titanium tetrahalide, TiX.sub.4, to afford a surface coating of Al-O-TiX.sub.y species. This surface coating has little effect on the surface properties of the inorganic oxide, yet provides a chemically reactive halide to other reagents. Diamines are an example of other reagents which react with the titanium-bonded halide, with one amino group firmly bound to the inorganic oxide via a strong titanium-nitrogen bond, and the other amino group subsequently reacting with a bifunctional reagent whose other functional moiety is available for covalent bonding to an enzyme.
Among the desirable properties of the resulting immobilized enzyme system is that the enzyme is held well away from the surface, thereby maximizing its opportunity to behave as it does in homogeneous reactions, and that the enzyme is covalently bonded to the support matrix, which generally imparts relatively high stability. An important difference between the support matrices and immobilized enzyme systems described here and those of the prior art is that drying of the titanated oxide is performed in an inert atmosphere, thereby preserving all titanium-halogen bonds. The prior art method assures that few, if any, titanium-halogen bonds will be present on the support matrix precursor.